bims-micesi Biomed News
on Mitotic cell signalling
Issue of 2022‒09‒04
thirteen papers selected by
Valentina Piano
Max Planck Institute of Molecular Physiology
  1. Cell cycle control of kinetochore assembly.
  2. Synchronization of HeLa Cells to Mitotic Subphases.
  3. The Four Causes: The Functional Architecture of Centromeres and Kinetochores.
  4. Cell Synchronization Techniques for Studying Mitosis.
  5. A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle.
  6. Processing DNA lesions during mitosis to prevent genomic instability.
  7. Synchronization of Saccharomyces cerevisiae Cells for Analysis of Progression Through the Cell Cycle.
  8. Synchronization of HeLa Cells to Various Interphases Including G1, S, and G2 Phases.
  9. Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes.
  10. Trip13 Depletion in Liver Cancer Induces a Lipogenic Response Contributing to Plin2-Dependent Mitotic Cell Death.
  11. Cell Cycle Progression and Synchronization: An Overview.
  12. Synchronization of Cultured Cells to G1, S, G2, and M Phases by Double Thymidine Block.
  13. Using a comprehensive approach to investigate the interaction between Kinesin-5/Eg5 and the microtubule.
  1. Nucleus. 2022 Dec;13(1): 208-220
    Cell cycle control of kinetochore assembly.
    Qianhua Dong, Fei Li.
      The kinetochore is a large proteinaceous structure assembled on the centromeres of chromosomes. The complex machinery links chromosomes to the mitotic spindle and is essential for accurate chromosome segregation during cell division. The kinetochore is composed of two submodules: the inner and outer kinetochore. The inner kinetochore is assembled on centromeric chromatin and persists with centromeres throughout the cell cycle. The outer kinetochore attaches microtubules to the inner kinetochore, and assembles only during mitosis. The review focuses on recent advances in our understanding of the mechanisms governing the proper assembly of the outer kinetochore during mitosis and highlights open questions for future investigation.
    Keywords:  CCAN; CENP-A; CENP-C; CENP-T; KMN; Kinetochore assembly; cell cycle; centromere; phosphorylation
    DOI:  https://doi.org/10.1080/19491034.2022.2115246
  2. Methods Mol Biol. 2022 ;2579 99-110
    Synchronization of HeLa Cells to Mitotic Subphases.
    Ping Wee, Richard C Wang, Zhixiang Wang.
      The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.
    Keywords:  Anaphase; Cell cycle; Fluorescence microscopy; Metaphase; Mitosis; Prometaphase; Synchronization; Telophase; Western blot
    DOI:  https://doi.org/10.1007/978-1-0716-2736-5_8
  3. Annu Rev Genet. 2022 Sep 02.
    The Four Causes: The Functional Architecture of Centromeres and Kinetochores.
    Andrew D McAinsh, Adele L Marston.
      Kinetochores are molecular machines that power chromosome segregation during the mitotic and meiotic cell divisions of all eukaryotes. Aristotle explains how we think we have knowledge of a thing only when we have grasped its cause. In our case, to gain understanding of the kinetochore, the four causes correspond to questions that we must ask: (a) What are the constituent parts, (b) how does it assemble, (c) what is the structure and arrangement, and (d) what is the function? Here we outline the current blueprint for the assembly of a kinetochore, how functions are mapped onto this architecture, and how this is shaped by the underlying pericentromeric chromatin. The view of the kinetochore that we present is possible because an almost complete parts list of the kinetochore is now available alongside recent advances using in vitro reconstitution, structural biology, and genomics. In many organisms, each kinetochore binds to multiple microtubules, and we propose a model for how this ensemble-level architecture is organized, drawing on key insights from the simple one microtubule-one kinetochore setup in budding yeast and innovations that enable meiotic chromosome segregation. Expected final online publication date for the Annual Review of Genetics, Volume 56 is November 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-genet-072820-034559
  4. Methods Mol Biol. 2022 ;2579 73-86
    Cell Synchronization Techniques for Studying Mitosis.
    Joanne D Hadfield, Sargun Sokhi, Gordon K Chan.
      Cell synchronization allows the examination of cell cycle progression. Nocodazole and other microtubule poisons have been used extensively to interfere with microtubule function and arrest cells in mitosis. Since microtubules are important for many cellular functions, alternative cell cycle synchronization techniques independent of microtubule inhibition are also used for synchronizing cells in mitosis. Here we describe using nocodazole, STLC, and combining thymidine block with MG132 to synchronize cells in mitosis. These inhibitors are reversible and mitotic cells can be released into the G1 phase synchronously. These techniques can be applied to both Western blot and timelapse imaging to study mitotic progression.
    Keywords:  Cell cycle synchronization; Double thymidine block; MG132; Mitotic arrest; Nocodazole; STLC; Timelapse imaging
    DOI:  https://doi.org/10.1007/978-1-0716-2736-5_6
  5. Proc Natl Acad Sci U S A. 2022 Sep 06. 119(36): e2206172119
    A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle.
    Scott Curran, Gautam Dey, Paul Rees, Paul Nurse.
      We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.
    Keywords:  cell cycle; genetics; yeast
    DOI:  https://doi.org/10.1073/pnas.2206172119
  6. Biochem Soc Trans. 2022 Aug 31. 50(4): 1105-1118
    Processing DNA lesions during mitosis to prevent genomic instability.
    Anastasia Audrey, Lauren de Haan, Marcel A T M van Vugt, H Rudolf de Boer.
      Failure of cells to process toxic double-strand breaks (DSBs) constitutes a major intrinsic source of genome instability, a hallmark of cancer. In contrast with interphase of the cell cycle, canonical repair pathways in response to DSBs are inactivated in mitosis. Although cell cycle checkpoints prevent transmission of DNA lesions into mitosis under physiological condition, cancer cells frequently display mitotic DNA lesions. In this review, we aim to provide an overview of how mitotic cells process lesions that escape checkpoint surveillance. We outline mechanisms that regulate the mitotic DNA damage response and the different types of lesions that are carried over to mitosis, with a focus on joint DNA molecules arising from under-replication and persistent recombination intermediates, as well as DNA catenanes. Additionally, we discuss the processing pathways that resolve each of these lesions in mitosis. Finally, we address the acute and long-term consequences of unresolved mitotic lesions on cellular fate and genome stability.
    Keywords:  DNA damage response; cell cycle; genome instability; genome integrity; mitosis
    DOI:  https://doi.org/10.1042/BST20220049
  7. Methods Mol Biol. 2022 ;2579 145-168
    Synchronization of Saccharomyces cerevisiae Cells for Analysis of Progression Through the Cell Cycle.
    Brianna L Greenwood, David T Stuart.
      The cell division cycle is a fundamental process required for proliferation of all living organisms. The eukaryotic cell cycle follows a basic template with an ordered series of events beginning with G1 (Gap1) phase, followed successively by S (Synthesis) phase, G2 (Gap 2) phase, and M-phase (Mitosis). The process is tightly regulated in response to signals from both the internal and external milieu. The budding yeast S. cerevisiae is an outstanding model for the study of the cell cycle and its regulatory process. The basic events and regulatory processes of the S. cerevisiae cell cycle are highly conserved with other eukaryotes. The organism grows rapidly in simple medium, has a sequenced annotated genome, well-established genetics, and is amenable to analysis by proteomics and microscopy. Additionally, a range of tools and techniques are available to generate cultures of S. cerevisiae that are homogenously arrested or captured at specific phases of the cell cycle and upon release from that arrest these can be used to monitor cell cycle events as the cells synchronously proceed through a division cycle. In this chapter, we describe a series of commonly used techniques that are used to generate synchronized populations of S. cerevisiae and provide an overview of methods that can be used to monitor the progression of the cells through the cell division cycle.
    Keywords:  Block-release; Cell cycle; Centrifugal elutriation; Hydroxyurea; Nocodazole; Synchronization; Yeast; cdc mutants; α-factor
    DOI:  https://doi.org/10.1007/978-1-0716-2736-5_12
  8. Methods Mol Biol. 2022 ;2579 87-97
    Synchronization of HeLa Cells to Various Interphases Including G1, S, and G2 Phases.
    Ping Wee, Richard C Wang, Zhixiang Wang.
      The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase, which allows the study of phase-specific cellular events. While interphase cells can be easily distinguished from mitotic cells by examining their chromosome morphology, it is much more difficult to separate and distinguish the interphases from each other. Here, we describe drug-derived protocols for synchronizing HeLa cells to various interphases of the cell cycle: G1 phase, S phase, and G2 phase. G1 phase synchronization is achieved through serum starvation, S phase synchronization is achieved through a double thymidine block, and G2 phase synchronization is achieved through the release of the double thymidine block followed by roscovitine treatment. Successful synchronization can be assessed using flow cytometry to examine the DNA content and Western blot to examine the expression of various cyclins.
    Keywords:  Cell cycle, synchronization, interphase, mitosis, G1 phase, S phase, G2 phase, flow cytometry, Western blot
    DOI:  https://doi.org/10.1007/978-1-0716-2736-5_7
  9. Nat Commun. 2022 Aug 27. 13(1): 5054
    Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes.
    Jiahong Li, Gerald J Shami, Ellie Cho, Boyin Liu, Eric Hanssen, Matthew W A Dixon, Leann Tilley.
      The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite's nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and -4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.
    DOI:  https://doi.org/10.1038/s41467-022-32579-4
  10. Adv Sci (Weinh). 2022 Aug 28. e2104291
    Trip13 Depletion in Liver Cancer Induces a Lipogenic Response Contributing to Plin2-Dependent Mitotic Cell Death.
    Marcos Rios Garcia, Bettina Meissburger, Jessica Chan, Roldan M de Guia, Frits Mattijssen, Stephanie Roessler, Andreas L Birkenfeld, Nathanael Raschzok, Fabien Riols, Janina Tokarz, Maude Giroud, Manuel Gil Lozano, Goetz Hartleben, Peter Nawroth, Mark Haid, Miguel López, Stephan Herzig, Mauricio Berriel Diaz.
      Aberrant energy metabolism and cell cycle regulation both critically contribute to malignant cell growth and both processes represent targets for anticancer therapy. It is shown here that depletion of the AAA+-ATPase thyroid hormone receptor interacting protein 13 (Trip13) results in mitotic cell death through a combined mechanism linking lipid metabolism to aberrant mitosis. Diminished Trip13 levels in hepatocellular carcinoma cells result in insulin-receptor-/Akt-pathway-dependent accumulation of lipid droplets, which act as functional acentriolar microtubule organizing centers disturbing mitotic spindle polarity. Specifically, the lipid-droplet-coating protein perilipin 2 (Plin2) is required for multipolar spindle formation, induction of DNA damage, and mitotic cell death. Plin2 expression in different tumor cells confers susceptibility to cell death induced by Trip13 depletion as well as treatment with paclitaxel, a spindle-interfering drug commonly used against different cancers. Thus, assessment of Plin2 levels enables the stratification of tumor responsiveness to mitosis-targeting drugs, including clinically approved paclitaxel and Trip13 inhibitors currently under development.
    Keywords:  MTOCs; Plin2; Trip13; hepatocellular carcinoma; lipogenesis; mitosis; spindle assembly checkpoint
    DOI:  https://doi.org/10.1002/advs.202104291
  11. Methods Mol Biol. 2022 ;2579 3-23
    Cell Cycle Progression and Synchronization: An Overview.
    Zhixiang Wang.
      The cell cycle is the series of events that take place in a cell that drives it to divide and produce two new daughter cells. Through more than 100 years of efforts by scientists, we now have a much clearer picture of cell cycle progression and its regulation. The typical cell cycle in eukaryotes is composed of the G1, S, G2, and M phases. The M phase is further divided into prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis. Cell cycle progression is mediated by cyclin-dependent kinases (Cdks) and their regulatory cyclin subunits. However, the driving force of cell cycle progression is growth factor-initiated signaling pathways that controls the activity of various Cdk-cyclin complexes. Most cellular events, including DNA duplication, gene transcription, protein translation, and post-translational modification of proteins, occur in a cell-cycle-dependent manner. To understand these cellular events and their underlying molecular mechanisms, it is desirable to have a population of cells that are traversing the cell cycle synchronously. This can be achieved through a process called cell synchronization. Many methods have been developed to synchronize cells to the various phases of the cell cycle. These methods could be classified into two groups: synchronization methods using chemical inhibitors and synchronization methods without using chemical inhibitors. All these methods have their own merits and shortcomings.
    Keywords:  Cdks; Cell cycle; G1 phase; G2 phase; M phase; S phase; Synchronization
    DOI:  https://doi.org/10.1007/978-1-0716-2736-5_1
  12. Methods Mol Biol. 2022 ;2579 61-71
    Synchronization of Cultured Cells to G1, S, G2, and M Phases by Double Thymidine Block.
    Richard C Wang, Zhixiang Wang.
      The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase. For many experiments, it is desirable to have a population of cells that are traversing the cell cycle synchronously, as it allows population-wide data to be collected rather than relying solely on single-cell experiments. While there are various drugs that can be used to arrest the cell at each specific phase of the cell cycle, they may cause undesired side effects. Here, we describe a protocol to synchronize cells to each cell cycle phase by using only one chemical: thymidine. Non-synchronized cells are synchronized to early S phase by a double thymidine block. The release of the double thymidine block allows the cells to progress through the cell cycle in a synchronized pace. By collecting the cells at various time intervals following the release of double thymidine block, we are able to harvest cells synchronized to the G2, M, and G1 phases. This synchronization can be assessed by various methods, including flow cytometry to examine the DNA content, Western blotting to examine the expression of various cell phase-specific markers, and microscopy to examine the morphology of the chromosome.
    Keywords:  Cell cycle; Flow cytometry; G1 phase; G2 phase; Interphase; Microscopy; Mitosis; S phase; Synchronization; Wester blotting
    DOI:  https://doi.org/10.1007/978-1-0716-2736-5_5
  13. Comput Struct Biotechnol J. 2022 ;20 4305-4314
    Using a comprehensive approach to investigate the interaction between Kinesin-5/Eg5 and the microtubule.
    Wenhan Guo, Shengjie Sun, Jason E Sanchez, Alan E Lopez-Hernandez, Tolulope A Ale, Jiawei Chen, Tanjina Afrin, Weihong Qiu, Yixin Xie, Lin Li.
      Kinesins are microtubule-based motor proteins that play important roles ranging from intracellular transport to cell division. Human Kinesin-5 (Eg5) is essential for mitotic spindle assembly during cell division. By combining molecular dynamics (MD) simulations with other multi-scale computational approaches, we systematically studied the interaction between Eg5 and the microtubule. We find the electrostatic feature on the motor domains of Eg5 provides attractive interactions to the microtubule. Additionally, the folding and binding energy analysis reveals that the Eg5 motor domain performs its functions best when in a weak acidic environment. Molecular dynamics analyses of hydrogen bonds and salt bridges demonstrate that, on the binding interfaces of Eg5 and the tubulin heterodimer, salt bridges play the most significant role in holding the complex. The salt bridge residues on the binding interface of Eg5 are mostly positive, while salt bridge residues on the binding interface of tubulin heterodimer are mostly negative. Such salt bridge residue distribution is consistent with electrostatic potential calculations. In contrast, the interface between α and β-tubulins is dominated by hydrogen bonds rather than salt bridges. Compared to the Eg5/α-tubulin interface, the Eg5/β-tubulin interface has a greater number of salt bridges and higher occupancy for salt bridges. This asymmetric salt bridge distribution may play a significant role in Eg5's directionality. The residues involved in hydrogen bonds and salt bridges are identified in this work and may be helpful for anticancer drug design.
    Keywords:  DelPhi; DelPhiForce; DelPhiPKa; Eg5; Electrostatic features; Hydrogen bonds; Kinesin-5; Microtubule; Molecular dynamic simulation; Salt bridges; Tubulin
    DOI:  https://doi.org/10.1016/j.csbj.2022.08.020
This page is being maintained by Thomas Krichel. It was last updated on 2023‒01‒06 at 16:10:44.